Discovery through Teamwork
Enlarge Photo
Photo courtesy of Michael W. W. Adams
The bacterium Caldicellulosiruptor bescii, named for the DOE BioEnergy Science Center (BESC).
Through centuries of evolution Nature has proved pretty ingenious in structuring the fibrous parts of plants to protect them against external threats, whether in the form of microbes, insects, or—for example—humans seeking to turn the plants into biofuels. Plant fiber is rich in sugars, and if we can get at those sugars, we can produce liquid fuels from them—that's what cellulosic biofuels are all about. But these plant sugars are enchained in molecules known as cellulose and hemicellulose, and these molecules are intertwined and protected in turn by a very tough substance known as lignin. It's this "tough customer"—lignin—that gives green plant stems their stiffness and is responsible for the hardness of wood. Scientists have a name for the problems posed by this tough customer: "recalcitrance." A major challenge in developing cellulosic biofuels is overcoming plant recalcitrance.
Because of recalcitrance, the process for producing cellulosic biofuels has until now been thought to require a pretreatment step. In pretreatment, the plant matter is immersed in very hot water, usually combined with acid or some other corrosive chemical, under high pressure. The point is to break the cellulose and hemicellulose loose from the lignin so that these molecules can be broken down into sugars by enzymes. But pretreatment poses two big problems. It adds to the cost—in fact, it's typically the most expensive step in the entire process. And it leaves trace chemicals that often get in the way of the subsequent processing by enzymes and, further down the pipeline, can inhibit the microbes whose job it is to ferment the sugars into fuel.
The DOE BioEnergy Science Center (BESC)—one of three DOE Bioenergy Research Centers (BRCs) established by the DOE Office of Science in 2007—has recently identified a microbe, isolated two decades ago from a hot spring in Russia, that performs what amounts to a minor miracle. This microbe simultaneously "solubilizes" or helps to dissolve all three major components of plant fiber: cellulose, hemicellulose, and lignin, without the need for pretreatment.
The identification of a microbe that can dissolve plant matter—in this case, switchgrass—without the need for pretreatment amounts to a major step forward. But there's more. BESC researchers have also developed a means of genetically modifying the microbe and have begun to coax it into producing fuel. The researchers have started out with hydrogen production but are aiming eventually at the production of liquid fuels. The ultimate goal would be a proof of concept for what is called "consolidated bioprocessing"—the use of a single organism or community of organisms to complete the process from end to end—to go from raw biomass to the production of liquid fuels in essentially a single step.
Interestingly, back when BESC was established in 2007, the researchers started with these two stated goals as their main focus: overcoming recalcitrance and developing consolidated bioprocessing. The latter goal, especially, seemed extraordinarily ambitious at the time. Now, thanks to a coordinated effort involving the University of Georgia, the DOE Joint Genome Institute (JGI), the DOE National Renewable Energy Laboratory, and several other institutions, they have managed to find a platform organism that could help make consolidated bioprocessing a reality.
“That's the wonderful thing about being part of a large scientific organization like BESC. You have access to both people and tools that as a single investigator you would never have.
” Michael W. W. Adams
The research was spearheaded by Michael W. W. Adams, a professor at the University of Georgia (UGA) and a principal investigator in BESC. In an effort to recruit the best talent nationwide, BESC, led by Oak Ridge National Laboratory, has enlisted a total of 18 other partner institutions, with researchers from universities, including UGA, national laboratories, private companies, and one nonprofit organization. All three BRCs—including also the Joint BioEnergy Institute led by Lawrence Berkeley National Laboratory and the Great Lakes Bioenergy Research Center led by the University of Wisconsin-Madison in partnership with Michigan State University—function as multidisciplinary, multi-institutional partnerships, with a strong emphasis on scientific teamwork and cooperation across institutional and disciplinary boundaries.
In formulating their research program, the leaders of the BESC team thought that the best candidates for consolidated bioprocessing would be thermophiles—or bacteria that thrive in extremely high-temperature environments. The bulk of research in this area had been done on a thermophile known as Clostridium thermocellum, which was capable of converting cellulose directly into ethanol. BESC researchers thought there could be other such microbes that might even do the job more effectively. The researchers even conducted a couple of field trips to hot springs in Yosemite in search of candidate microbes.
Adams, a nationally recognized specialist in thermophiles, found his microbe via the scientific literature. He was scanning for thermophiles that were known to be "cellulolytic," that is, having the capacity to break down cellulose. He found literature on a microbe that had been isolated from a hot spring in the far east of Russia in the 1990s. He ordered samples of the microbe from a German repository and cultured it in his laboratory. He discovered that the microbe, which thrives at temperatures around 78°C, did appear indeed to be highly cellulolytic.
At this point, BESC asked the JGI to sequence the microbe's genome. Today such genomic sequencing is typically the first step to understanding what an organism is and how it works. Established as part of the Human Genome Project, JGI now almost exclusively sequences microbes and plants with relevance to energy and the environment. It provides sequencing on a priority basis to the three DOE Bioenergy Research Centers. It's one key to the ability of the BRCs to make rapid research progress.
"That's the wonderful thing about being part of a large scientific organization like BESC," said Adams. "You have access to both people and tools that as a single investigator you would never have."
The sequence was completed in 2009. As a result of the sequencing and subsequent analysis, the researchers determined that the microbe had been classified as part of the wrong genus. This gave them the opportunity to reclassify the bacterium—and in the process to rename it as a new species. Adams thought, why not name the bug for BESC? The scientific name is a mouthful: Caldicellulosiruptor bescii. The abbreviated name is easier: C.bescii (pronounced "SEE BESKY EYE").
Enlarge Photo
Photo courtesy of Michael W. W. Adams
Michael W. W. Adams of the DOE BioEnergy Science Center and the University of Georgia led the research.
The sequencing was critical, but sequencing alone was insufficient to understand the capabilities of the organism. It would be necessary to systematically study the action of the microbe on plant biomass. This required tools and expertise beyond what Adams had at his disposal in his UGA laboratory. Again, the multi-institutional structure of BESC permitted Adams to draw on the instrumentation and expertise of several BESC partner institutions, including DOE's National Renewal Energy Laboratory, which has special facilities and instruments for studying biomass processing.
It was through this latter research that Adams discovered that the C.bescii can solubilize all three major components of raw switchgrass without pretreatment. (The researchers analyzed what was left over of the plant matter after the C.bescii did its work. They expected to find mostly lignin, but instead they found cellulose, hemicellulose, and lignin in comparable quantities—which meant that the bug had solubilized all three.) According to Adams, it is not that the microbe digests lignin per se, but rather that it is so effective at solubilizing cellulose and hemicellulose that the lignin may simply fall away in pieces. But the ability to get around the plant's tough customer without the aid of pretreatment is a very special talent.
Subsequently a University of Georgia-based team of BESC researchers led by UGA professor Janet Westpheling has developed a method of genetically modifying the microbe. The initial modification (deletion of a single gene) suppressed lactate production by the bacterium and increased the microbe's output of hydrogen gas. Further modifications aimed at producing liquid fuels are planned, with the aim of showing proof of concept for consolidated bioprocessing with this microbe.
Adams is quick to point out that research on C.bescii and its genus is at a very early stage, and C.bescii has its limitations. At present it can solubilize only about a third of a switchgrass sample in a single round. Adams speculates that other members of the Caldicellulosiruptor genus may prove to be more effective. But to be able to overcome recalcitrance, absent pretreatment, with the possibility of moving from plant biomass to fuel via a single microbe is a very promising development—and shows the power of large-scale coordinated science efforts to tackle a research challenge.
--Patrick Glynn, DOE Office of Science, [email protected]
Funding
DOE Office of Science, Office of Biological and Environmental Research
Publication
Irina Kataeva, Marcus B. Foston, Sung-Jae Yang, Sivakumar Pattathil, Ajaya K. Biswal, Farris L. Poole II, Mirko Basen, Amanda M. Rhaesa, Tina P. Thomas, Parastoo Azadi, Victor Olman, Trina D. Saffold, Kyle E. Mohler, Derrick L. Lewis, Crissa Doeppke, Yining Zeng, Timothy J. Tschaplinski, William S. York, Mark Davis, Debra Mohnen, Ying Xu, Art J. Ragauskas, Shi-You Ding, Robert M. Kelly, Michael G. Hahn and Michael W. W. Adams, "Carbohydrate and lignin are simultaneously solubilized from unpretreated witchgrass by microbial action at high temperature," Energy and Environmental Science, published online May 17, 2013.
Related Publications
Minseok Cha, Daehwan Chung, James G Elkins, Adam M Guss, and Janet Westpheling, "Metabolic engineering of Caldicellulosiruptor bescii yields increased hydrogen production from lignocellulosic biomass," Biotechnology for Biofuels 6, 85 (2013).
Daehwan Chung, Joel Farkas, and Janet Westpheling, "Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement," Biotechnology for Biofuels 6, 82 (2013).
Daehwan Chung, Minseok Cha, Joel Farkas, and Janet Westpheling, "Construction of a Stable Replicating Shuttle Vector for Caldicellulosiruptor Species: Use for Extending Genetic Methodologies to Other Members of This Genus," PLOS One, published online May 3, 2012
Sung-Jae Yang, Irina Kataeva, Juergen Wiegel, Yanbin Yin, Phuongan Dam, Ying Xu, Janet Westpheling, and Michael W. W. Adams, "Classification of 'Anaerocellum thermophilum' strain DSM 6725 as Caldicellulosiruptor bescii sp. nov.," International Journal of Systematic and Evolutionary Microbiology 60, 2011 (2010).
Irina A. Kataeva, Sung-Jae Yang, Phuongan Dam, Farris L. Poole II, Yanbin Yin, Fengfeng Zhou, Wen-chi Chou, Ying Xu, Lynne Goodwin, David R. Sims, John C. Detter, Loren J. Hauser, Janet Westpheling, and Michael W. W. Adams, "Genome Sequence of the Anaerobic, Thermophilic, and Cellulolytic Bacterium 'Anaerocellum thermophilum' DSM 6725," Journal of Bacteriology 191, 3760 (2009).
Sung-Jae Yang, Irina Kataeva, Scott D. Hamilton-Brehm, Nancy L. Engle, Timothy J. Tschaplinski, Crissa Doeppke, Mark Davis, Janet Westpheling, and Michael W. W. Adams, "Efficient degradation of lignocellulosic plant biomass without pretreatment by the thermophilic anaerobe, Anaerocellum thermophilum DMS 6725" Applied Environmental Microbiology 75, 4762 (2009).
